Supercharged Fluorescent Protein-Apoferritin Cocrystals for Lighting Applications

The application of fluorescent proteins (FPs) in optoelectronics is hindered by the need for effective protocols to stabilize them under device preparation and operational conditions. Factors such as high temperatures, irradiation, and organic solvent exposure contribute to the denaturation of FPs, resulting in a low device performance. Herein, we focus on addressing the photoinduced heat generation associated with FP motion and rapid heat transfer. This leads to device temperatures of approximately 65 °C, causing FP-denaturation and a subsequent loss of device functionality. We present a FP stabilization strategy involving the integration of electrostatically self-assembled FP-apoferritin cocrystals within a silicone-based color down-converting filter. Three key achievements characterize this approach: (i) an engineering strategy to design positively supercharged FPs (+22) without compromising photoluminescence and thermal stability compared to their native form, (ii) a carefully developed crystallization protocol resulting in highly emissive cocrystals that retain the essential photoluminescence features of the FPs, and (iii) a strong reduction of the device’s working temperature to 40 °C, leading to a 40-fold increase in Bio-HLEDs stability compared to reference devices.


Methods
Thermocycler-based modulated scanning fluorimetry Modulated Scanning Fluorimetry was performed as described in Svilenov et al. 1 The Thermocycler CFX96 Touch Real-time PCR System (Bio-Rad) was employed to perform MSF measurements.One standard program composed of heating and cooling cycles ranging from 25 °C to 99 °C was used to measure the progressive loss of fluorescence and the irreversible unfolding of the FPs studied in this work.The samples were heated 5 °C/sec and held for 1 min at the temperature peak, followed by a recovery period of 5 min at 25 °C.Due to the high sensitivity of the Thermocycler detector and high QY of the FPs used in this study, only 1 μM of FPs were added per well to avoid saturation.The thermograms were buffer-subtracted and normalized by the highest fluorescence read-out of each sample.Data analysis was performed using Origin 2019 (OriginLab Corporation, Northampton, MA, USA).Mean values and standard deviations of quintuplicate were calculated and plotted.Melting curves were obtained plotting the fluorescence values obtained at peak temperatures, while non-reversibility curves were obtained plotting the fluorescence values obtained at 25°C.The nonreversibility temperatures (T nr ) areas were determined via the integration tool available in the software.

Computational methods
RMSD comparisons: the CA atoms from the conserved glycines in fluorescent protein 2 and the CA from the two flanking residues per chromophore were used for RMSD comparison of every relaxation against the best relaxed structure per protein population.
Cavity detection and Cavity Volume calculations: for Cerulean, Cerulean-32, mGL and scmGL protein structures, the ParKVFinder software 3 was used with default values for whole protein exploration: whole protein mode, low resolution mode, prove in of 1.4 Å, probe out of 4.0 Å, volume cutoff of 5.0 Å 3 , and removal distance of 2.4 Å.The volumes in cavities of interest per protein were added for comparison purposes.ParKVFinder usage, along with cavity representations and structure superpositions, were carried out on PyMol 2.5.0.

Dynamic light scattering
The hydrodynamic diameter (D h ) of the assemblies was measured using a Malvern Instruments DLS device (Zetasizer Nano ZS Series) with a 4 mW He-Ne gas laser at a wavelength of 633 nm and an avalanche photodiode detector at an angle of 173°.All experiments were carried at room temperature.PMMA cuvettes were used for the size measurements.Zetasizer software (Malvern Instruments) was used to obtain the particle size distributions: 0.1 mg mL -1 of aFt (with a final concentration of 0.25 mM NaCl) dissolved in buffer (20 mM Tris (pH 7.5)) was titrated with different mGL or scmGL concentrations (0.05, 0.1, 1, 10 mg mL -1 ) to reach the desired ratio (no dilution correction was done as the total addition did not exceed 5 % of sample volume), which was finally titrated with 5 M NaCl to disassemble the complex.

Cryogenic transmission electron microscopy
The cryo-TEM images were collected using JEM 3200FSC field emission microscope (JEOL) operated at 300 kV in bright field mode with an Omega-type zero-loss energy filter.The images were acquired with Gatan Digital Micrograph software while the specimen temperature was maintained at -187 °C.The cryo-TEM samples were prepared by placing 3 μL aqueous dispersion of the sample on a 200-mesh Lacey carbon film on Copper TEM Grids (agar scientific) and plunge-frozen into liquid ethane using Leica grid plunger with 3 s blotting time under 100 % humidity.The grids with vitrified sample solution were maintained at liquid nitrogen temperature and then cryo-transferred to the microscope.The TEM grids were plasma cleaned (20 seconds oxygen plasma flash using a Gatan Solarus).Images were further processed using ImageJ software.

Small-angle X-ray scattering
The SAXS samples were measured using the Xenocs Xeuss 3.0 C device equipped with a GeniX 3D Cu microfocus source (wavelength λ = 1.542Å) and EIGER2 R 1M hybrid pixel detector at a sample-to-detector distance of 0.6 m.One-dimensional SAXS data was obtained by azimuthally averaging the 2D scattering data and the magnitude of the scattering vector q is given by q = 4π sinθ / λ, where 2θ is the scattering angle.For all the measurements, the scattering vector q was calibrated using a silver behenate standard and the 2D scattering data were converted into SAXS curves by azimuthal averaging.The samples were sealed in 1 or 1.5 mm glass capillaries (Hilgenberg GmbH) that has limited scattering in the measured q region.

Optical microscopy
The Zeiss Axiovert A1 inverted microscope was used to perform imaging through optical microscopy.To prevent distortion of the crystal habit, a chamber-like area was created using double-sided tape on all four sides to hold the sample (3 µL) between the glass slide and coverslip.

Confocal fluorescence microscopy and photobleaching
The confocal fluorescence and brightfield imaging of the crystals was done using a spinning disk confocal microscope (Nikon Ti-E with Crest Optics X-Light V3 scanner and Hamamatsu Orca Flash 4.0LT camera) with photobleaching capabilities (Gataca iLas2) and 60x/1.2Wobjective lens.The system was controlled using Micro-Manager.Small volumes (3 uL) of the crystal samples (aFt and aFt-scmGL) in buffer were placed between two precision cover glasses (Thorlabs CG15KH) separated from each other using a double sided tape spacer and imaged under identical conditions.Fluorescence images were excited using 470 nm laser (LDI Laser Diode Illuminator) at 1% power level with exposure time of 5 ms.Z-stacks were collected similarly at 500 nm steps.The brightfield images were collected using the same microscope in the transmitted light mode using a red LED (Thorlabs).The selected area photobleaching was done using 405 nm laser (Coherent OBIS 405 nm LX 100mW) at 50% power by raster scanning the beam to form the desired area in ca.500 ms defined in the software plugin (Gataca Modular).
The confocal fluorescence and brightfield imaging of the silicone based devices (fresh and post-mortem) was done using a point scanning laser confocal microscope (Zeiss LSM710 on Examiner frame).Fluorescence was excited using using 488 nm laser at 2.5% power and emission was collected above 493 nm with 2.5x/0.06objective lens (low magnification images) and 40x/1.1Wobjective lens (high magnification images).Fresh sample was the silicone resin mixed with crystals cured on a glass coverslip.The post-morten sample was a bulk piece of moded resin containing crystals.

Photophysical characterization of solutions and coatings
The aFt-scmGL with 50 mM NaCl crystal samples were prepared as described above.The crystals were incubated in the refrigerator for 24 h for sedimenting the crystals.After the incubation, the supernatant was replaced with fresh buffer (20 mM Tris with pH 7.5).For measurements, the UV-Vis and fluorescence spectra were measured using a Cytation 3 plate reader (BioTek) using 96-well plates.Alternatively, absorption spectra were acquired with a UV-vis spectrometer UV-2600 (Shimadzu), using a wavelength range 200-800 nm, scan speed medium, threshold 0.01 and a slit width of 2.0. was determined by relative measurement.The molar extinction coefficient was measured using the "alkali-denatured" method, 4 in which scmGL was denatured in 0.5 M NaOH.Under these conditions, the chromophore was converted to a GFP-like chromophore characterized by an extinction coefficient of 44,000 M−1cm−1 at 446 nm.The molar extinction coefficient was calculated based on the absorbance spectra of the denatured and native scmGL.The molecular brightness results from the product of photoluminescence quantum yield and the molar absorption extinction coefficient.The photophysical studies were carried out using a FS5 Spectrofluorometer (Edinburgh Instruments) with the SC-10 module for solid samples, the SC-30 Integrating Sphere to determine ϕ, and the time-correlated single photon-counting or TCSPC (64.3 ps pulse width) module to determine τ.All τ were recorded with an excitation bandpass fixed at 450 nm (τ 450 ) and at the emission maximum.The data was then adjusted to a mono-or bi-exponential decay fit using Origin Software.To calculate the average lifetime for each FP-coating, the following equation was used ; <  > 0 = where a i (λ) is the amplitude fractions and τ i are the lifetimes.The measurements were performed at room temperature.All the lifetimes recorded were measured with a TCSPC.